Gastrin is a peptide hormone which occurs in two mature forms, tetratriacontagastrin (G34) and heptadecagastrin (G17), and is synthesized and secreted by specialized cells, G cells, that are located in the stomach antrum. In gastrin-producing cells, these gastrin hormones are posttranslationally processed from a common precursor molecule termed “preprogastrin” containing a signal peptide. The signal peptide “pre” is removed in the endoplasmic reticulum of the cell, resulting in the “progastrin” peptide, which is in turn further processed in the cell to yield the mature gastrins G34 and G17, before secretion into the bloodstream (Dickinson 1991). (The full citations for the references cited herein are provided in the Reference Section preceding the Claims): Both mature forms of G34 and G17 are amidated at their carboxy-terminal end (—NH2). In humans, multiple forms of G17 have been found resulting from differential processing of the precursor molecule, each of which may have different biological activities (Dickinson 1995 and Ciccotosto et al. 1995). In the posttranslational processing of gastrin, it is the “mature” carboxy-amidated form that binds to a specific cell receptor, the so-called CCK-B/gastrin receptor, via the carboxy terminus of the peptide (Kopin et al. 1992).
The gastrin hormones are secreted into the circulating blood and bind to specific cells in the stomach, namely, enterochromaffin-like (ECL) cells and parietal cells, that indirectly or directly affect stomach acid output. Historically, both gastrin hormones have been associated with the stimulation of gastric acid secretion (Edkins, J. S. 1905). In recent years, evidence has accumulated showing that gastrin also acts as a trophic factor within the gastrointestinal tract (Johnson, L. 1997) and that it promotes the growth of gastrointestinal cancers (Watson et al. 1989, Dickinson, C. J. 1995), as well as nongastrointestinal cancers, including small cell carcinoma of the lung (Rehfeld et al. 1989).
Several types of tumors, including colorectal, stomach, pancreatic and hepatocellular adenocarcinomas possess CCK-B/gastrin receptors in their plasma membranes and the tumor cells respond to gastrin- with powerful cellular proliferation (Rehfeld, J. F. 1972, Upp et al. 1989 and Watson et al. 1993). Elevated plasma levels of total gastrin occur in patients with colorectal cancers, and, in particular, increased amounts of the hormone precursor progastrin have been detected in many colorectal tumors using gastrin antisera (Ciccotosto et al. 1995). More recently, it has been discovered that many of these cancer cells also secrete gastrin and thus effect an autonomous proliferative pathway (Van-Solinge et al. 1993, Nemeth et al. 1993 and Seva et al. 1994).
The peptide hormones G17 and G34 bind to the CCK-B/gastrin receptors on the cell membranes of normal cells. However, it has been found that G17, but not G34, stimulates the growth of gastrin-dependent cancer cells. Serum-associated G17, in particular, has the potential to stimulate the growth of colorectal tumors in an .endocrine manner mediated by CCK-B/gastrin receptors in tumor cells (Watson et al. 1993). G17 is particularly implicated in stimulating the growth of colorectal adenocarcinomas due to a possible increased affinity for the CCK-B/gastrin receptors on the tumor cells, as compared to other gastrin hormone species (Rehfeld 1972 and 1993). The CCK-B/gastrin receptors were found to be expressed in a high affinity form on 56.7% of human primary colorectal tumors (Upp et al. 1989).
Numerous studies have shown that, in addition to being able to respond to exogenous endocrine gastrin, human gastric and colorectal tumors produce gastrin and its precursors (Ciccotosto et al., 1995; Finley et al., 1993; Kochman et al., 1992; Nemeth et al., 1993; Van Solinge et al., 1993), thus effecting an autocrine growth stimulatory pathway. Gastrin production in tumor cells differs from that of endocrine G cells. Specifically, those tumor cells contain a high proportion of the precursor progastrin along with a lower concentration of mature peptides. This abnormal ratio is postulated to be due to constitutive unregulated release of gastrin combined with a limited activity of peptidylglycine α-amidating monooxygenase (Ciccotosto et al., 1995; Kelly, 1985). Thus, the unregulated release of gastrin leads to the abnormal production and secretion of different molecular forms of the hormone. Specifically, colon carcinoma cells do not efficiently process progastrin resulting in less conversion of precursor gastrin to the mature peptides and, thus, produce mostly incomplete or aberrant gastrins, (Dickinson 1993 and Rehfeld et al. 1993). In addition, the increased gastrin level in colorectal tumors is, in part, attributed to the aberrant expression of the gastrin gene in the colorectal tumor cells (Hoosein et al. 1990, Baldwin et al. 1992 and Finley et al. 1993). Gastrin-like peptides have been identified in such cells (Hoosein et al. 1988, Watson et al. 1991 and Finley et al. 1993), and were confirmed to be precursor gastrin species (Van-Solinge et al. 1993 and Nemeth et al. 1993).
The presence of amidated G17 (G17-NH2) in some colorectal cancers (Ciccotosto et al., 1995; Van Solinge et al., 1993) demonstrates that some tumors retain an intact processing pathway, as gastrin amidation only occurs in secretory granules (Varro et al, 1994). Endogenously produced gastrin also acts as an autocrine growth factor, since the basal growth of a colorectal cell line was shown to be inhibited by an anti-gastrin antibody (Hoosein et al., 1988). This was confirmed in a second study in which Northern blot analysis revealed gastrin mRNA in the same cell lines and radioimmunoassay revealed gastrin-like immunoreactivity in cell culture supernatant (Hoosein et al., 1990).
Gastrin peptides also possess paracrine roles (Watson et al., 1991b) which was confirmed (Finley et al., 1993) in experiments showing gastrin immunoreactivity more predominant in subpopulations of malignant colorectal mucosal cells.
When G17 binds to its receptor a G17/receptor complex is formed which stimulates cell growth by way of secondary messengers for regulating cell function (Ullrich et al. 1990). The binding of G17 to the CCK-B/gastrin receptor leads to activation of phosphatidylinositol breakdown, the protein kinase C activation with a resultant increase in intracellular calcium ion concentration, and the induction of c-fos and c-jun protooncogenes via the mitogen-activated protein kinase, which has been implicated in the regulation of cell proliferation (Tadisco et al. 1995). Additionally, gastrin binding to the CCK-B/gastrin receptor has been associated with the subsequent increase in phosphorylation by a tyrosine kinase, the pp125FADK (focal adhesion kinase), which may also have a role in the transmission of mitogenic signals (Tanaguchi et al. 1994).
Colorectal cancer remains a formidable disease to treat, as only minor improvements in survival have been obtained in recent years. Surgery is an effective treatment of the primary disease, but it is ineffectual against residual occult disease, which is frequently present. Radiation therapy post-surgery is generally recommended for patients with rectal cancers to reduce the risks of recurrence of the disease. Chemotherapy with 5-fluorouracil (5-FU) has been the most traditional effective therapy following surgery in patients with more advanced colorectal cancers. However, 5-FU therapy has been shown to be only of marginal benefit to the patient, since 5-FU is highly toxic and the therapy is costly and does not appear, alone or in combination with other cytotoxic drugs, to significantly prolong survival. In most instances, occult or inoperable colorectal tumors do not respond well to chemotherapy or radiation, and new treatments are needed to supplement present procedures.
Recently, several studies have shown that adjuvant combination chemotherapy with 5-FU and Leucovorin improves the efficacy of 5-FU in patients with advanced colorectal cancer. Leucovorin is a folic acid -derivative, also known as folinic acid, Citrovorum factor, or 5-formyl-5,6,7,8,-tetrahydrofolic acid. The studies show that in Dukes' stage C patients, 5-FU/Leucovorin combination therapy may reduce mortality by 10 to 15% (Moertel, 1994). In the same patient group, combined intravenous and intraperitoneal therapy with 5-FU/leucovorin resulted in a non-significant trend to disease-free survival and overall survival advantage (Scheithauer et al., 1995). In advanced disease, the same drug combination may give rise to a survival advantage (Taylor, 1993), which has been shown to be 13.5 months of median survival in the” combination group compared to 7.5 months in 5-FU-treated patients (Petrioli et al., 1995). However, this combination chemotherapy is not without significant morbidity and causes deleterious side effects including stomatitis, diarrhea and myelosuppression (Mahood et al., 1991; Erlichman et al., 1988; Pietnelli et al., 1989), making quality of life an issue, especially in patients with advanced disease.
A number of high affinity CCK-B/gastrin receptor antagonists have been evaluated therapeutically both in vitro and in vivo in a number of experimental gastrointestinal cancers. For example, proglumide, a glutamic acid derivative (Seva et al. 190; Harrison et al. 1990 and Watson et al. 1991a); Benzotript, an N-acyl derivative of tryptophan; L-365,260, a derivative of Aspercillin (Bock et al. 1989); and CI-988, a molecule that mimics the C-terminal pentapeptide sequence of CCK (Hughes et al. 1990), have been shown to effectively neutralize the effects of exogenous gastrin on gastrointestinal tumor growth both in vitro and in vivo (Watson et al. and Romani et al. 1994). However, these antagonists have severe toxic side effects and lack specificity, as they block the action of all potential ligands of the receptor such as G34 and CCK in normal cells. Recently, highly potent and selective CCK-B/gastrin receptor antagonists such as YM022 (Yuki et al., 1997) and YF476 (Takinaini et al., 1997) have been also described.
Proglumide and Benzotript have been widely assessed in preclinical studies. The main problem with these compounds is their lack of potency, with relatively high concentrations required to displace G17 (Watson et al., 1992a; Watson et al., 1992b).
Despite this, proglumide and Benzotript inhibited the basal and gastrin-stimulated proliferation of a number of cell lines (Seva et al., 1990; Watson et al., 1991a). In addition, proglumide increased the survival of xenograft mice bearing the gastrin-sensitive mouse colon tumor MC26 to 39 days in the treated animals from 25 days in the control animals.
Due to the low specificity of this class of gastrin antagonizing agents for the gastrin/CCK-B receptor, the inhibition of growth is also thought to be induced by a gastrin-receptor-independent action. Moreover, the cellular receptors which recognize and bind the gastrin do not bind all the inhibitors tested (Seva et al. 1994). Thus, if complete inhibition of gastrin binding to the receptor does not occur in the autocrine growth cascade, the gastrin antagonists may be unable to block this mechanism of tumor growth promotion.
Thus, novel therapeutic approaches are needed both as modalities in their own right and for combination strategies with chemotherapy. Combined treatments offer the possibilities of enhancing the therapeutic index and/or reducing the dose of chemotherapy required, thereby limiting the disadvantageous side effects.
A therapeutic method of selectively immunologically neutralizing the biological activity of the gastrin hormone would provide an effective means of controlling or preventing the pathologic changes resulting from excessive gastrin hormone production associated with colorectal cancers.
Coassigned U.S. Pat. Nos. 5,023,077; 5,468,494; 5,607,676; 5,609,870 and 5,622,702 disclose immunogens and immunogenic compositions useful for controlling G17 and G34 levels in a patient by generating anti-gastrin antibodies and also disclose the use of such compositions for the treatment of gastric and duodenal ulcers and gastrin-induced cancers. The present invention concerns the use of the anti-G17 immunogens and immunogenic compositions disclosed in U.S. Pat. Nos. 5,023,077; 5,468,494; 5,607,676; 5,609,870 and 5,662,702 in a combination therapy with chemotherapeutic agents for treating gastrin-dependent colorectal cancers.
The method of cancer therapy described herein has several advantages over present colorectal cancer treatment methods. The anti-G17 immunization, in combination with chemotherapeutic agents such as 5-FU and Leucovorin, increases the therapeutic effects in controlling or inhibiting colorectal tumor growth over chemotherapy alone.